Mergapto folic acid and process of



Patented July 24,1951 7 2 55 553 UNITED STATES PATENT OFFICE MERGABTO some ACID AND PROCESS OF PREPARING. SAME.

Thomas Samuel Gardner, Rutherford; and Edward Wenis, Leonia, N. J'., assignnrs to Hofimann-Lai Roche Ii1c., Nutley, N.,J., a corporationof New Jersey No Drawing. ApplicationApril 15, 1948,

Serial No. 21,304

7 Glaimsi. (Cl. 260-4515) The presentinvention relates to a new synthesis duce N [4- [(2 --.lower alkylmercapto-i-hyof folic acid, and to new intermediates useful droxy-fi-pteridyl) methyll-amino} benzoyl-l in the preparation of this compound. glutamic acid or 2-1ower alkylm'ercapto-z-des- Folic acid, also knownas'pteroylglutamic acid, amino-folic acid (VD. Compound (VI-l is then has" been" obtained synthetically by reacting to- 5 converted by ammonolysis to folic acid (VII).

gether 2,4,5-triamino-6-hydroxy pyrimidine, p- Ihstead of starting with 2-mercapto-4,5-diaininobenzoyl-l-(+)-glutamie acid and 2,3-diamino-fi hydrox-y-pyrimidine (I) there can be bromopropionaldehyde, or a halopyruvic aldeemployedasa starting material a z-lower'alkylhyde, such as chloropyruvic aldehyde. mercapto-Afi-di'amino-6-hydroxy-pyrimidine; as

Thepresent invention providesa newmethod 10 for example z methylmercapto-4,5-diarnino6 for producing folic acid. The methodremployed hydroxy-pyrimidine. This is subjected to the lnsthe above equation R stands for a. lower by"the. .2-lo.weralkylmercapto-compoundscorrealkylLgtoup, such as methyl, ethyl, propy1, isospending to. (II), (III) and. (V) are obtained. propyl',.and.the. likehandxstands for a halogen y. a t h a W y p o-45- such as chlorine. diamino-fi-hydroxy-Dyrimidine, compound (VI) Thus 2-me1 captq- 4 5- diamino- 6 hydroxy- 40 iS directly obtained, and the step of alkylating pyrimidine formate. (I), which per se. is a known o p und (V)- eliminated. compound. andcan be prepared by methods de- Compou ds (I (V) and (VI), and scribed in; the literature, is condensed. with dith e pon ng" 2-1ower alkylme eflpt dehydroxy-acetonein thepresence of anoxidizing v s" are mp u ds a d f m part of agent, such as hydrazine to form. 2-mercapto- 11 on.

4-hydroxy-6 hydroxymethyl-pteridine (II) which The followingexampleswill serve to illustrate is. halogenated, as. for example by thionyl chloour invention.

ride, to produce 2-mercapto-4-hydroxy-6-haloflillmiflle' I genomethyI-pteridine (III). The latter is. then Sixt 40 r L condensed withwp-aminobenzoyl-IJ -g1utamic ?g 1 g i ggi gg i gggggg (IV) 1 form" *'4 in 250 cc. of water, and 2'7 g. of sodium acetate roxy's'ptendyb methyl]ammoi-benzoyll-gwand 15g. of acetic acid added. The suspension tamic acid. or 2-mercapto-2. desamino-iolic acid w h ated up to 100C. A solution (a) of 22.80 (vl'which is. alkylated by reacting (V) with a g'... of 85%- hydrazine was mixed with 250 cc. of lower alkyl ha11de.,such. as methyl: iodide, to: prowateranda solution (b) of 30.3 g; of d'ihydroxyarated out and when the reaction was completeds 3 r:

the mixture was cooled, solids filtered offand washed with water, alcohol, and ether.

1.116 Twenty grams of the 2-mercapto-4-hydroxy- 6-hydroxymethyl-pteridine so obtainedweresus-,

pended in 200 cc. of thionylichlorideqandi heated .3

under reflux for one hour with stirring 'When the reaction was completed as indicated by cessation of the evolution of hydrochloric acid, the ex:

cess thionyl chloride was removed in vacuum .i, .The'

2-mercapto -4-hydroxy-6-chloromethyl-pteridine 4 46.5 grams of dihydroxyacetone at 100 C. for two hours in the presence of 91 grams of sodium acetate, 21 grams of acetic acid and 19.5 grams of hydrazine hydrate. On cooling, 12.5 grams of .cru de 5- 2 .-methylmercapto 4 hydroxy; 6; -hydroxy- -methylepteridinefwereobtained. The 12.5 grams of pteridine compound were heated under reflux with 100 ml. of thionyl chloride for 4 hours and the excess thionyl chloride was then removed by vacuum distillation and displacement by toluene.

The "Z-methyImercapto-I-hydroxy 6 chloromethyl-pteridine thus prepared was heated at 50 ,C. forFfour-hours in ethylene glycol solution with 42.6 grams of p-aminobenzoyl-l-(+)-glutamic acid. The reaction mixture was poured into 1 .liter of cold water and the pH adjusted to 4 with dilute'hydrochloric acid. After standing for 16 hours the material was filtered off and dried after thus obtained was then suspended in toluene and the toluene removed in vacuum in order to eliminatefinal tracesof thionyl chloride. Eight grams of 2-mercapto-4-'hydroxy6-chloe romethyl-pteridine and 25.2 :g. of p-aminobenzoyl-l-(+) glutamic acid were dissolved in 250 cc. of ethylene glycol and heatedto- 50 .C.- for four hours; Eighty -five cc. of this reaction mixture was mixed with 20 g. of methyl iodide and cc. of ethanol and allowed to stand overnight at 25 C. This yields a solution containing 2 -.methylmercaptO-Z-desamino follic. acid. r

Fifty-five cc. of the, reaction mixturewas saturated with ammonia gas at 25 C. and then heated on a water bath for three hours under reflux.

The reaction mixture was poured into water, the

pI-I adjusted to 4 and the precipitate was filtered off. Ten grams of the crude product obtained in this way was dissolved in 100 cc. of ammonia solution, the pH adjusted to 4,"10g.of diatoma ceous earth were added as an absorbent and the Example 2 eluate, f olic Fifty-five cc. of the solution of Z-rnethyImercapto-Z-desamino-folic acid asv obtained in Example 1 was mixed with 100' cc. of concentrated ammonia solution and heated on a water bath for three hours. It was then poured into water, the pH adjusted to 4 and filtered. The precipitate wasdissolved in 10 cc. of ammonia solution, ad justedto pH 4, and 10 g. of diatomaceous earth were added as an absorbent. The mixture was stirred for a few minutes and filtered. .The'ad sorbate,was washed with alcohol and then eluted as described-in Example l to-yield folic acid.

By following the procedure of'Examples 1 and 2, but employing 2-methylmercapto-4,5-diamino 6 hydroxy-pyrimidine as a starting material there are obtained 2-methylmercapto-4-hydroxy6hy droxymethyl-pteridine, 2 methylmercapto-4- hydroxy-6-chloromethyl-pteridine, and -2 n'lethylmer captc-2-desamino-folic acid, in that order, no alkylation step, of course, being required; The 2-methylmercapto-2-desamino-folic acid, is converted by ammonolysis into folic acid .in the same manner as described in the examples. The following examples is illustrative of this procedure.

' Example 3 washing with water.

Yield 8.05 grams of crude 2-methylmercapto2-desamino-folic acid. 250, milligrams, of -the Z-methylmercapto-2- desamino -folic acid; 'thus prepared were refluxed with concentrated-ammonia solution (-1 00;, ml.), for 4 hours.. The solution was diluted to -lliter, grams of diatomaceous earth added, ;stirred and filtered after two hours. The filter cake was washed with ml...of alcohol three. times and eluted ,three times with 100' m1. of,.the. eluting solvent described in Example 1. .The eluted material was concentrated under vacuum ,to dryness-toyieldfolic acid. 7 Weclaim: 1. The process whic comprises condensing a compound of the groupconsisting of 2-'mercapto- 4,5-diamino-6-hydroxy pyrimidine and 2-lower alkylmercapto J 4,5'- diamino-6-hydroxy pyrimidines with dihydroxy-acetone in the presence of an oxidizing agent to form the corresponding mercaptoai-hydroxy-G-hydroxymethyl-pteridine, halogenating the last mentioned compound to form the corresponding mercapto-4-hydroxy-6- halogeno-methyl-pteridine, and condensing the latter with p-am-inobenzoyl-l-(+) -glutamic acid to form the'corresponding acid, which can be' wherein is selected from the group consisting of hydrogen and lower alkyl. 2-. The process which comprises condensing 2- mercaptQ-4,5-diamino "6 hydroxy pyrimidine formats with dihydroxy acetpne in the presence of hydrazine, chlorinating the condensation prod: not formed with thionyl chloride to form; 2- mercapto 4 hydroxy-6 chloromethyl pteridine. and condensing the latter compoundwith', pamino benzoyl l f-l-j) -fglutamicjacid tojf orin N: [4 {I 2, mercapto 4 -'hydroxy 6 pteridyl): methyl]-amino}-benzoyll-glutamic acid. 3. .A process which; comprises condensing methylmercapto 4,5 diamino-6-hydr0xyfpyrimr; dine with dihydroxy-acetone the presence a: hydrazine, chlorinating the condensation prod; not with thionyl chloride to form 2 -'metliylmerf capto 4 hydroxyrfi chloromethyl-pteridine and condensing the latter compound with p-ammo benzoyl-l-(+) -glutamic acid to form N-[4{['( 2 methylrnercapto-4- hydroxy-6-pteridyl) -methyll amino}-benzoyll-glutamic acid. I 4. N [4- {-[(2-mercapto-4-hydroxy6-pteri dyl) -methyl] -amino}-benzoy1l -glutamic acid: j '5. N'-[4-{-[(2 -lower alkylm'er'ca'pto-4 hydroxy '5 6-pteridy1) -methy1]-amino}-benzoy1] glutamic acid.

6. N [4-{ (2-methy1mercapto 4 hydroxy-G- pteridyl) methyl] amino} benzoyl] -g1uta,mic

acid.

'7. A compound selected from the group consisting of N [4 {[(2 mercaptol-hydroxy-fipteridy1)'- methyl] amino} benzoyl] --glutamic acid and N-[4-{ (2-1ower a1ky1mercapt0-4-hydroxy 6 pteridy1)-- methyl] -amino}-benzoy1]- glutamic acid.

THOMAS SAMUEL GARDNER. EDWARD WENIS.

No references cited. 

7. A COMPOUND SELECTED FROM THE GROUP CONSISTIN GOF N-(4-(((2-MERCAPTO-4-HYDROXY-6PTERIDYL)-METHYL)-AMINO)-BENZOYL)-GLUTAMIC ACID AND N-(4-(((2-LOWER ALKYLMERCAPTO-4-HYDROXY-6-PTERIDYL)-METHYL)-AMINO)-BENZOYL)GLUTAMIC ACID. 